实时荧光定量PCR检测鸡传染性贫血病毒方法的建立

建立了Taqman实时荧光定量PCR方法检测鸡传染性贫血病毒(CAV)。选取CAV病毒的序列设计引物和探针。以梯度稀释的含有CAV目的扩增片段的质粒作为标准品,进行定量PCR反应以确定检测灵敏度。2.0×105～2.0×102个拷贝,4个数量级的范围内定量PCR有"S"型扩增曲线,检测灵敏度为200个拷贝。根据病毒拷贝数与定量反应Ct值的关系,绘制了标准曲线。该方法具有特异性,对传染性支气管炎病毒、传染性法氏囊病毒、禽白血病病毒和马立克病毒核酸都没有扩增反应。采用实时荧光定量PCR方法检测试验感染鸡的肝脏、脾脏、肾脏、胸腺、法氏囊、泄殖腔棉拭子等样品,所有检测样品都有"S"型扩增曲线,且荧光信号值高。实时定量PCR检测CAV的方法,灵敏度高,特异性好,可以进行定量分析,在禽病的快速检测上具有重要意义。     

家禽免疫抑制病液相芯片检测方法的建立

本研究针对传染性法氏囊病毒、鸡传染性贫血病毒、J亚型禽白血病病毒、马立克病毒1型基因组、新城疫病毒、禽流感病毒的特异性核酸序列,分别设计了6种病毒的特异性引物、通用扩增引物,及荧光编码微球包被用特异性探针。借助全新的通用扩增技术实现6种病毒核酸同时扩增,再与高通量的液相芯片检测相结合,进而实现4 h内6种病毒的快速准确检测。该方法创新性地将液相芯片技术GMPLex引入到动物检疫行业,突破了DNA病毒、RNA病毒不能同时检测及一次PCR不能完成检测的瓶颈,实现了一管一次PCR反应同时检测6种病毒,缩短检验周期,节约检验成本。     

荧光定量PCR检测鸡马立克氏病血清1型病毒方法的建立及应用

为了建立一种快速的鸡马立克氏病血清1型病毒(MDV-1)病原检测方法,试验采用针对MDV-1基因序列保守区域设计1对特异性引物和1条特异性的探针,通过构建重组阳性标准质粒的方法,构建重组质粒作为阳性标准品,建立了检测MDV-1核酸的荧光定量PCR方法。优化反应体系和条件后进行特异性、敏感性、重复性试验。结果显示,该检测方法特异性强,与其它禽源病毒如新城疫病毒(NDV)、禽流感病毒(AIV)、传染性支气管炎病毒(IBV)、传染性法氏囊病病毒(IBDV)、鸡传染性贫血病毒(CIAV)和J亚群禽白血病病毒(ALV-J)均不发生交叉反应;该方法可检测到4.6×101拷贝/L的病毒核酸,与常规PCR相比,敏感性高100倍;重复性试验的变异系数小于2%。研究结果表明所建立的Real-time PCR检测方法特异性强、灵敏度高、可重复性好,可用于MDV-1的定量检测。     

非洲猪瘟病毒常规PCR及Real-time PCR检测方法的建立

根据非洲猪瘟病毒(African swine fever virus,ASFV)P72基因的核苷酸序列,设计并合成引物以及荧光标记的TaqMan探针,以含P72基因的重组质粒作为阳性模板,用于常规PCR和Real-time PCR方法的建立,结果表明常规PCR的检测灵敏度是600个拷贝的病毒核酸分子,Real-time PCR的检测灵敏度是20个拷贝的病毒核酸分子,两种PCR检测方法均具有特异性强、简单快速的优点。可以用于出入境检验检疫部门对非洲猪瘟病毒的快速检测。     

应用聚合酶链反应检测鸡传染性贫血病毒

根据鸡传染性贫血病毒（CAV）Cux－1株的基因序列,设计了一对引物,用这对引物对三株CAV的核酸模板进行PCR扩增,结果均能特异性地扩增出420bp的片段,但对其它5种禽病病原体核酸模板的扩增,结果均为阴性,该PCR能检出10fg鸡传染性贫血病毒DNA模板。     

东方马脑脊髓炎病毒PCR-DHPLC检测方法的建立与初步应用

为建立检测东方马脑脊髓炎病毒(EEEV)的方法,本研究利用PCR结合变性高效液相色谱技术(DHPLC),针对EEEV NS1基因,设计特异性引物,通过优化反应条件,对核酸扩增产物采用DHPLC进行检测分析,核酸扩增产物形成DHPLC特征峰图,建立了EEEV的PCR-DHPLC快速检测方法。结果显示该方法与其他6种常见马病病毒均无交叉反应,未检测到其他马病病毒的阳性吸收峰,具有较强的特异性;对梯度稀释的标准阳性模板的检测限可达到10拷贝/μL;利用本研究建立的方法与荧光定量RT-PCR对30份临床样品进行检测,两者符合率达100%。本研究建立的PCR-DHPLC法具有特异、敏感、快速、重复性好等优点,可以用于EEE的病原学诊断及流行病学调查。     

基于便携式荧光定量PCR仪的鸡传染性贫血病毒检测方法的建立及应用

本研究参照Gen Bank中鸡传染性贫血病毒基因组序列,设计特异引物和TaqM an探针,通过优化反应条件,建立了能检测鸡传染性贫血病毒的荧光PCR方法,并验证该方法的特异性、敏感性和重复性。结果表明,该方法检测鸡传染性贫血病毒灵敏度可达2.69TCID50/100μL,该法对禽呼肠孤病毒(AR V)、马立克氏病病毒(MDV)、禽白血病病毒(ALV)、禽网状内皮组织增生病病毒(REV)和传染性法氏囊病病毒(IBDV)的检测结果均为阴性。本试验建立的TaqM an荧光定量PCR检测方法可对鸡传染性贫血病毒进行快速鉴别诊断,为鸡传染性贫血的诊断及防控净化工作奠定了基础。     

实时荧光定量RT-PCR检测禽白血病病毒方法的建立与应用

建立了TaqMan实时荧光定量RT-PCR方法检测禽白血病病毒(ALV)。选取ALV病毒的LTR序列设计引物和探针,以梯度稀释的含有ALV目的扩增片段的质粒作为标准品,进行定量PCR反应以确定检测灵敏度。阳性标准品在3.0×102~3.0×107个拷贝共6个数量级的范围内,定量PCR反应有"S"型扩增曲线,检测灵敏度最低为30个拷贝。根据病毒拷贝数与定量反应Ct值的关系,绘制了标准曲线。该方法具有特异性,对新城疫病毒、禽流感病毒、传染性支气管炎病毒、传染性囊病病毒、鸡传染性贫血病毒和马立克病病毒核酸都没有扩增反应。实时定量PCR检测ALV的方法,灵敏度高,特异性好,可以进行定量分析,在禽病的快速检测上具有重要意义。     

应用荧光定量RT-PCR检测J亚群禽白血病病毒

针对J亚群禽白血病病毒（ALV-J）基因序列保守区域设计一对特异性引物和一条特异性探针,通过构建重组阳性标准质粒作为阳性标准品,建立了检测ALV-J核酸的荧光定量PCR方法。优化反应体系和条件后进行特异性、敏感性、重复性试验。结果显示,该检测方法特异性强,与其它禽源病毒如A亚群禽白血病病毒（ALV-A）、B亚群禽白血病病毒（ALV-B）、新城疫病毒（ NDV）、禽流感病毒（AIV）、鸡传染性贫血病毒（CIAV）和马立克病病毒（MDV）均不发生交叉反应;该方法可检测到3.2×102拷贝/μL的病毒核酸,与常规RT-PCR相比,敏感性高100倍;重复性试验的变异系数小于2%。研究结果表明,建立的Real-time RT-PCR 检测方法特异性强、灵敏度高、可重复性好,可用于ALV-J的定量检测。     

荧光定量RT-PCR检测鸡传染性支气管炎病毒方法的建立及应用

为了建立一种快速的新城疫病毒(NDV)病原检测方法,试验针对鸡传染性支气管炎病毒(IBV)基因序列保守区域设计1对特异性引物和1条特异性探针,通过构建重组阳性标准质粒的方法建立了检测IBV核酸的荧光定量PCR方法,优化反应体系和反应条件后进行特异性、敏感性、重复性试验。结果表明:该检测方法特异性强,与其他禽源病毒如NDV、传染性喉气管炎病毒(ILTV)、传染性法氏囊病毒(IBDV)、禽流感病毒(AIV)、马立克病毒(MDV)均不发生交叉反应;该方法可检测到3.1×101拷贝/μL的病毒核酸,比常规RT-PCR的敏感性高100倍;重复性试验的变异系数小于2%。说明研究建立的Real-time RT-PCR检测方法特异性强、灵敏度高、重复性好,可用于IBV的定量检测。     

绵羊肺腺瘤病毒实时荧光定量PCR检测方法的建立

为建立检测绵羊肺腺瘤病毒(Jaagsiekte sheep retrovirus,JSRV)实时荧光定量PCR(Real-time qPCR)方法,根据外源性JSRV-NM株env基因序列,选其保守序列作为目的片段,设计引物和TaqMan探针,以自然病例的肺肿瘤组织基因组DNA为模板,经PCR扩增目的基因、克隆,重组质粒鉴定,并严格定量后,梯度稀释作为阳性标准品,优化反应条件进行Real-time qPCR扩增,获得的标准曲线为:Y=-3.308X+47.848,线性相关系数为0.991;Ct值变异系数小,并且灵敏度高,初步建立了检测JSRV前病毒DNA的Real-time qPCR方法。应用该方法对不同来源(A、B、C、D、E组)的绵羊外周血及其他组织样品进行测定其前病毒载量。结果显示B组和C组外周血白细胞、肺脏、肺门淋巴结以及鼻液中检测均为阳性,并发现前病毒DNA的载量在肺脏中明显高于外周血白细胞;D组虽未发现有绵羊肺腺瘤(SPA)临床症状,但在肺门淋巴结里可以检测到;E组中1只绵羊的肺脏也检出低拷贝数的前病毒DNA,而在A组中检测结果均为阴性。本研究对检测未知羊群JSRV感染程度及研究SPA流行病学等均有重要意义。     

化脓隐秘杆菌TaqMan实时荧光定量PCR检测方法的建立

本试验旨在建立检测化脓隐秘杆菌(Arcanobacterium pyogenes,A.pyogenes)特异、灵敏的TaqMan实时荧光定量PCR检测方法。根据GenBank公布的化脓隐秘杆菌溶血素(pyolysin,PLO)基因高保守序列,设计特异性引物和探针建立检测体系,用于化脓隐秘杆菌的快速检测,并对该方法的特异性和灵敏度进行检测。结果显示,本试验建立的TaqMan实时荧光定量PCR方法仅对化脓隐秘杆菌的检测结果为阳性;该方法最低检测DNA浓度为77.6 fg,最低检测细菌浓度为63 CFU/mL。采用本研究建立的方法检测23份林麝临床病例样品,共鉴定出16株化脓隐秘杆菌,与API Coryne生化鉴定方法的结果相同。本研究为化脓隐秘杆菌的检测提供了一种灵敏、特异、快速的检测方法,其可用于化脓隐秘杆菌的诊断和流行病学调查。     

Establishment of TaqMan Real-time PCR Assay for Detection of Arcanobacterium pyogenes

The study was aimed to establish a specific and sensitive TaqMan Real-time PCR assay for detection of Arcanobacterium pyogenes (A.pyogenes).Based on the conservative sequence of pyolysin (PLO) gene of A.pyogenes published in GenBank, specific primers and TaqMan probes were designed. The TaqMan Real-time PCR assay was established, and the specificity and sensitivity were tested.The specificity test results showed that only A.pyogenes exhibited typical curves.The detection sensitivity of this assay was 77.6 fg genomic DNA per 20 μL reaction, and 63 CFU/mL for pure cultures.16 out of 23 clinical samples were positive detected by the TaqMan Real-time PCR assay, which were consistent with API Coryne identification.The TaqMan Real-time PCR assay developed in this study was specific and sensitive for detection of A.pyogenes, and it could be used for identification and epidemiological investigation of A.pyogenes.     

Establishment and Application of A Double TaqMan MGB Real-time PCR Assay for Detection of Canine Distemper Virus and Canine Parvovirus

This study was aimed to establish a double TaqMan MGB Real-time PCR assay to simultaneously and specifically detect canine distemper virus (CDV) and canine parvovirus (CPV) in one reaction.Two pairs of specific primers for CDV and CPV,along with two TaqMan MGB probes for each virus were designed in the assay basing on CDV H gene and CPV VP2 gene sequences.The specificity,sensitivity and repetition of the double TaqMan MGB Real-time PCR assay were tested,and 48 samples taken from clinic suspicious CDV and CPV infected canines had been testified by the established double TaqMan MGB Real-time PCR.The results indicated that the doulde TaqMan MGB Real-time PCR assay was successfully established,and the number of standard curve correlation (R²) of CDV and CPV were 0.997 and 0.993,respectively.The specificity of the double TaqMan MGB Real-time PCR assay revealed that amplifications were showed on CDV and CPV samples,but other pathogens and negative controls had no amplifications;The sensitivity of CDV and CPV were both 10 copies/μL.Meanwhile,14 CDV positive samples,19 CPV positive samples and 4 CDV/CPV double positive samples were detected,which were consistent with the results of the sequencing.Therefore,the established double TaqMan MGB Real-time PCR assay had high sensitivity,specificity and flux accurate quantitative,which could be applied to clinical CDV/CPV infection each periods.     

伪狂犬病病毒野毒株与gE基因缺失疫苗株TaqMan实时荧光定量PCR鉴别方法的建立

为实现对伪狂犬病病毒（pseudorabies virus,PRV）野毒株与gE基因缺失疫苗株的快速、敏感、特异的鉴别诊断,本试验针对PRV gD和gE基因设计了2套特异性引物和TaqMan探针,建立了PRV野毒株与gE基因缺失疫苗株的TaqMan实时荧光定量PCR鉴别方法,对引物和探针浓度、退火温度等进行了优化,对方法进行敏感性、特异性、重复性试验,并进行临床样品检测。结果显示,建立的针对gD、gE基因的TaqMan实时荧光定量PCR方法线性相关系数（R²）分别为0.996和0.980,均呈良好的线性关系;检测限分别为39.4和12.1拷贝/μL;与圆环病毒2型、猪瘟病毒、猪繁殖与呼吸综合征病毒均无交叉反应;重复性试验结果显示,针对gD基因的批内和批间变异系数分别为1.43%~1.86%、1.10%~2.07%,针对gE基因的批内和批间变异系数分别为0.98%~1.41%、1.12%~1.86%。应用建立的TaqMan实时荧光定量PCR与普通PCR分别对11份临床疑似感染样品进行检测,阳性率分别为36.4%和27.3%。结果表明,该方法敏感性高、特异性强、重复性好,可作为伪狂犬病病毒野毒株与gE基因缺失疫苗株的早期鉴别诊断和定量检测的有效手段。     

Establishment of TaqMan Real-time PCR Method for Detection of Duck Poxvirus

To establishment a TaqMan Real-time PCR method for detection of duck poxvirus (DPV),we cloned the P4b gene of DPV.The specific primers and probe were designed according to the nucleotide sequence of avipoxvirus available in GenBank.Recombinant plasmid pMD-DPV-P4b was employed as positive standard template for Real-time PCR.By optimization of reaction conditions,a TaqMan Real-time PCR method for detection of DPV was established.The results of specificity test proved this method had no cross-react with other waterfowl vial agents and poxviruses including avian influenza virus,duck flavivirus,duck hepatitis virus,Newcastle disease virus,duck entertitis virus,goose parvovirus,goatpox virus and fowlpox virus.The detection limit of the assay was 1.29×10² copies/μL of viral DNA,which was 100 times higher than that of the routine PCR.Reproducibility test showed that the CVs of intra assay and inter assay were both less than 2%.Above results supported that the assay was suitable for the detection of DPV very well.     

鸭痘病毒TaqMan荧光定量PCR检测方法的建立

为建立检测鸭痘病毒的TaqMan荧光定量PCR方法,本试验克隆了鸭痘病毒P4b基因,构建重组质粒pMD-DPV-P4b,并将其作为标准阳性模板。参照GenBank收录的禽痘病毒P4b基因设计合成1对特异性引物及与该引物相匹配的特异探针。以定量的10倍系列稀释的质粒pMD-DPV-P4b为标准品,通过对反应条件进行优化,建立了一种检测鸭痘病毒的TaqMan荧光定量PCR方法。结果显示,该方法与禽流感病毒、鸭黄病毒、鸭肝炎病毒、新城疫病毒、鸭瘟病毒和小鹅瘟病毒等其他水禽病毒,以及山羊痘病毒和鸡痘病毒等其他痘病毒均无交叉反应,特异性好。该方法最低检测限为1.29×10²拷贝/μL,比普通PCR检测方法高100倍。组内和组间变异系数均小于2%。结果表明,本试验所建立方法具有灵敏、特异、安全、快速的特点,适用于鸭痘病毒的检测。     

弓形虫Real-time PCR检测方法的建立和初步应用

根据已发表的弓形虫529 bp高拷贝序列,设计特异性引物和探针,建立TaqMan探针法的实时荧光定量PCR检测方法。将该方法用于360份临床样本的检测,并与常规PCR和环介导等温扩增(LAMP)技术进行对比分析。结果表明:Real-time PCR检测的阳性率(11.11%)高于LAMP(7.5%)和常规PCR(3.61%)。采用本实验室建立的Real-time PCR,对收集于不同地区4种动物的750份临床样本检测发现,不同种类动物血液样本中均可检测到弓形虫感染,其中羊的感染率最高(17.8%),猪次之(4.22%),牛较低(1.98%),犬最低(1.37%)。     

Establishment of a Duplex TaqMan Real-time PCR Method for Detection of Bovine Viral Diarrhea Virus Types 1 and 2

【Objective】 This study was aimed to establish a duplex TaqMan Real-time PCR method for rapid detection of Bovine viral diarrhea virus types 1(BVDV1) and 2(BVDV2).【Method】 Specific primers were designed based on the 5'-non-coding region of 95 strains of BVDV1 and BVDV2 in GenBank.The positive plasmids containing the target fragments of BVDV1 and BVDV2 were constructed.The reaction conditions were optimized, the standard curve was constructed, the specificity, sensitivity and reproducibility of the duplex TaqMan Real-time PCR method were tested, and the established duplex TaqMan Real-time PCR assay was used to detect the clinical samples collected from Jilin province.【Result】 The results showed that the optimal annealing temperature of the duplex TaqMan Real-time PCR was 57.0 ℃, the optimum primer concentration was 0.5 μmol/L, and the optimum probe concentration was 0.3 μmol/L.The standard curves of BVDV1 and BVDV2 were Y=-3.54X+37.36 (R²=0.990) and Y=-3.18X+35.95 (R²=0.997), respectively.There was no specific amplification of Infectious bovine rhinotracheitis virus (IBRV), Bovine respiratory syncytial virus (BRSV) and Bovine parainfluenza virus type 3 (BPIV3), with intra- and inter-batch CV less than 3%, and the lower limit of detection was 10 copies/μL.The results of clinical samples showed that the overall positive rate was 23.1% (36/156), of which 17.9% (28/156) were positive for BVDV1 and 5.1% (8/156) were positive for BVDV2.【Conclusion】 In this study, a duplex TaqMan Real-time PCR method was established, which could identify BVDV types 1 and 2 simultaneously, quickly and accurately, and provided technical support for the prevention, control and purification of BVDV.